Our research is devoted to the study of a fundamental membrane function of mammalian cells, namely the transport of macromolecules at the cell surface. One of our interests is to develop procedures that will enhance the cellular uptake of macromolecules that are ordinarily only poorly transported. By conjugating a fragment of poly(lysine) of 9,000 Mr to horseradish proxidase at a ratio of 1:1, the cellular uptake of active enzyme by cultured fibroblasts has been increased 1,000-fold. This finding has prompted us to use poly(lysine) as a carrier for anticancer drugs. By conjugating methotrexate (MTX) to a poly(lysine) of 70,000 Mr at a ratio of 13:1, the cellular uptake of MTX is increased 20-\to 40-fold in normal CHO cells, and 200-\to 400-fold in CHO mutants defective in drug transport. In these cells, resistance due to defective MTX transport can be overcome by using MTX-poly(lysine), but not by using MTX-poly(D-lysine). The latter conjugate is ineffective because it does not release active drug inside cells. These drug carriers are now used as models in three lines of investigation pursuing the following goals: (1)\to define optimal drug-linkages for the intracellular release of drugs delivered by macromolecular carriers. An\acid-sensitive linkage that cleaves spontaneously in an acidic intracellular compartment has been developed. (2)\To isolate and characterize mutants which are sensitive to MTX, but resistant to MTX-poly(lysine), and which are therefore defective in their ability to process a foreign macromolecule. One such mutant has been found to be pleiotropically defective in endocytosis. (3)\To develop procedures that will allow a selective targeting of macromolecular drug carriers to tumor cells. Methotrexate-carrying human serum albumin, complexed with anti-human serum albumin IgG, has been used to kill selectively two Fc-Receptor positive tumor cells lines in vitro. (A)